Cell transplantation has over the last two decades emerged as a promising approach for restoration of function in neurodegenerative diseases, in particular Parkinson's and Huntington's disease. Clinical trials have so far focused on the use of implants of embryonic mesencephalic tissue containing already fate-committed dopaminergic neuroblasts with the capacity to develop into fully mature dopamine neurons in their new location in the host brain. A major limitation of the fetal cell transplantation procedure is the low survival rate of the grafted neurons (in the range of 5-20%) which makes it difficult to obtain sufficient cells for grafting in patients. Currently, mesencephalic fragments from at least 6-8 embryos are needed for transplantation in one Parkinson's disease patient. Moreover, the ethical, practical and safety issues associated with the use of tissue from aborted human fetuses are problematic, and severely restrict the possibility for applying the procedure outside highly specialized centers.
It was recently demonstrated that immature neural progenitor cells with multipotent properties, called neural stem cells (NSCs), can be isolated from both the developing and adult CNS. Neural stem cells are multipotent cells which can be differentiated into any type of neural cell, including neurons, astrocytes, glia and oligodendrocytes. The successful propagation of mammalian neural stem cells (NSC) in culture, first reported by Reynolds and Weiss (1992), has opened up hitherto unforeseen opportunities in the field of neural transplantation and, therefore, harvesting these cells from donated adult human tissue is of great interest.
However, it would be advantageous to isolate the NSCs from the other cells in the brain or enrich them such that the purest population of multipotent cells possible can be obtained. One favored strategy for cell isolation is the identification of target epitopes on the surface of NSCs accessible to monoclonal antibodies. These antibodies can then be selectively tagged, e.g., with a fluorescent label, whereupon selecting for the tagged antibody results in selection of the cell on which it is bound. If the target molecule is expressed only by the desired cell type, very high levels of enrichment are possible. The problem with this strategy is that of identifying the target epitopes which will isolate the NSCs, allow for specificity of attachment and will not activate cellular processes.
The identification and enrichment using specific cell surface markers has been used previously in the isolation of another type of stem cell, neural crest stem cells (NCSC's) (Anderson and Stemple, 1998, U.S. Pat. No. 5,824,489). However, the use of the method in the isolation of neural stem cells (NSCs) has been slow to develop, possibly due to the difficulty in identifying NSC-specific markers. Uchida et al., 2000, describes a method for the isolation of NSCs using a specific epitope. In this work, the authors restrict their definition of human neural stem cells to those cells within the human brain which are CD133+/CD34− (and CD24−/lo) and describe the use of this marker profile to isolate NSCs. They also state that the CD133+/CD34− (and CD24−/lo) fraction alone contains neural stem cells because neurospheres could not be generated from the CD133− fraction. However, there are a number of problems and inconsistencies with this method. For example, the authors state that neurospheres could not be generated from the CD133− population. However, the population and method of enriching may have produced variability in frequency of neurosphere initiating cells resulting from such manipulation. It is likely that the process of mincing, enzymatically-dissociating, and sorting the cells twice, increased levels of damage to constituent cells, leaving a number of the cells non-viable.
Embryonic stem cells have been shown to be useful for transplantation treatment of a number of diseases. Since 1987, about 250 patients with advanced Parkinson's Disease have received transplants of mesencephalic dopamine neurons, obtained from 6-9 week old cadaver embryos at several centers in Europe and America. There is now convincing data to show that embryonic human nigral neurons, taken at a stage of development when they have started to express their dopaminergic phenotype, can survive, integrate and function over a long time in the human brain (i.e. in a tissue environment with an ongoing disease process). Embryonic stem cells are very primitive, non-neuronal cells which can be induced to differentiate into neural progenitor cells by the treatment with specific morphogens. Thus, there is reason to believe that neural progenitors or neural stem cells (NSCs) could be used for the same purpose. For example, neural stem cells were shown to be useful for the treatment of hypoxic-ischemic (HI) brain injury (stroke). When NSCs were injected into mice brains subjected to focal HI injury, they appeared to integrate appropriately into the degenerating central nervous system (CNS), and showed robust engraftment and foreign gene expression within the region of HI injury. They also appeared to have migrated preferentially to the site of ischemia, experienced limited proliferation, and differentiated into the neural cells that were lost to injury, trying to repopulate the damaged brain area. Therefore, the transplantation of exogenous NSCs may, in fact, augment a natural self repair process in which the damaged CNS “attempts” to mobilize its own pool of stem cells. Providing additional NSCs and trophic factors may optimize this response (Park, KI; 2000, Yonsei Med J, Dec;41(6):825-35). Therefore, NSCs may provide a novel approach to reconstituting brains damaged by HI brain injury as well as Parkinson's disease and other neurodegenerative disorders.
Because NSCs appear to be excellent candidates for restorative cell replacement and gene transfer therapies, and could eventually offer a powerful alternative to primary fetal CNS tissue in clinical transplantation protocols, methods for the successful isolation from adult brain are needed.